Schisandrin B for the treatment of osteosarcoma

Abstract

This invention relates to the use of schisandrin B in the treatment of osteosarcoma. The invention reveals that schisandrin B has a remarkable effect in treating osteosarcoma, inhibiting the growth, proliferation, migration, and invasion of osteosarcoma cells, and inducing osteosarcoma cell apoptosis; it also exhibits a synergistic effect when used in combination with drugs such as cisplatin, improving therapeutic efficacy and reducing toxic side effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals. Specifically, this invention relates to the use of schisandrin B in the preparation of pharmaceuticals. This invention also relates to pharmaceutical compositions and pharmaceutical combination products for the treatment of osteosarcoma. Background Technology

[0002] Osteosarcoma (OS) is the most common primary malignant bone tumor, originating from bone-forming mesenchymal stem cells. It primarily affects the metaphysis of long bones and is characterized by its high recurrence rate, metastasis rate, and aggressiveness. The highest incidence is in young adults and children under 20 years of age (average age 18 years), followed by the elderly over 60 years of age. The initial clinical manifestation is pain at the tumor site, which progresses from intermittent to persistent. A palpable, significantly swollen mass is present at the site of pain, and the hardness of the mass varies depending on the degree of ossification. As the disease progresses, patients experience pain-induced limping and limited joint movement. In my country, osteosarcoma remains a leading cause of death among malignant tumors in children and adolescents. Currently, the main clinical treatment for osteosarcoma is a combination of radical tumor resection and neoadjuvant chemotherapy. Commonly used chemotherapy drugs include methotrexate (MTX), adriamycin (ADM), cisplatin (DDP), and ifosfamide (IFO), which can be administered as single drugs or in combination, depending on the treatment needs. While adjuvant chemotherapy can have a significant therapeutic effect to some extent, chemotherapy drugs have high resistance rates and significant side effects. The 5-year survival rate for children and young adults with localized disease reaches 78%, but it is less than 20% for patients with metastasis or recurrence. Commonly used chemotherapy regimens include doxorubicin + cisplatin, mesna + adriamycin + ifosfamide + dacarbazine, and methotrexate + adriamycin + cisplatin, among others. Cisplatin is the first-line drug in osteosarcoma chemotherapy. Clinically, after low-dose cisplatin treatment of osteosarcoma, osteosarcoma cells can quickly repair damage, escape apoptosis, and return to their original high-proliferative state; only high-dose cisplatin can produce a strong anti-tumor effect. However, long-term use of high-dose cisplatin not only leads to drug resistance but also causes serious side effects, primarily manifesting as nephrotoxicity, digestive system damage, and hematopoietic system disorders. Other side effects include ototoxicity, neurotoxicity, and immune system suppression. Therefore, to reduce tumor drug resistance and drug side effects, new synergistic drug therapy strategies should be developed. Numerous clinical trials have demonstrated that traditional Chinese medicine (TCM) not only has anti-tumor effects but also possesses holistic benefits that many chemotherapy drugs lack. Combining chemotherapy drugs with TCM in anti-tumor treatment can not only reduce the side effects of chemotherapy drugs but also effectively improve patient survival and prolong their lifespan.

[0003] Schisandrin B (Sch B) is one of the active ingredients of Schisandra chinensis, a plant in the Magnoliaceae family. Its structure is as follows:

[0004]

[0005] Schisandrin B possesses various activities and plays an important role in hepatoprotection, antioxidation, anti-aging, and anti-tumor activity. While widely used to inhibit tumor growth, no literature reports its role in the treatment of osteosarcoma. Summary of the Invention

[0006] This invention is the first to discover that schisandrin B has a good effect in treating osteosarcoma. It can inhibit the growth activity, migration and invasion of osteosarcoma cells. When used in combination with cisplatin, it can improve the effect of inhibiting tumor growth and reducing toxic side effects.

[0007] Therefore, a first aspect of the present invention is to provide the use of schisandrin B in the preparation of a medicament for the treatment of osteosarcoma.

[0008] This invention has discovered that schisandrin B has a good effect in treating osteosarcoma. It can greatly inhibit the growth, migration and invasion of osteosarcoma cells, and induce osteosarcoma cell apoptosis by enhancing autophagy.

[0009] Therefore, preferably, the drug is used to inhibit the proliferation and growth of osteosarcoma cells and / or inhibit the migration and invasion of osteosarcoma cells and / or induce osteosarcoma cell apoptosis. More preferably, the induction of osteosarcoma cell apoptosis is achieved by enhancing osteosarcoma cell autophagy.

[0010] A second aspect of the present invention is to provide a pharmaceutical composition for treating osteosarcoma, said pharmaceutical composition comprising schisandrin B.

[0011] In this invention, the drug and the drug composition may contain pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients include one or more of the following: pharmaceutically acceptable carriers, excipients, diluents, fillers, disintegrants, binders, emulsifiers, lubricants, flow aids, flavoring agents, odorants, and coloring agents.

[0012] The pharmaceutical compositions described in this invention can be prepared as oral or non-oral medications in the form of tablets, pills, capsules, granules, powders, liquids, emulsions, suspensions, ointments, injections, and skin patches using common formulation techniques.

[0013] The pharmaceutical compositions of the present invention can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0014] In the pharmaceuticals and pharmaceutical compositions of the present invention, the amount of schisandrin B can be from 0.01% to 50% of the total weight of the composition, preferably from 0.1% to 10%, and more preferably from 0.5% to 5%.

[0015] For adult patients, schisandrin B can be administered orally or non-orally in a single dose of 0.001–500 mg, once daily or divided into several doses. It should be noted that the dosage may be adjusted according to the type of disease, age, weight, and symptoms of the patient.

[0016] The schisandrin B of the present invention can be used alone or in combination with other therapeutic agents. When used in combination with other therapeutic agents, the combination therapy can provide a synergistic or additive effect. The synergistic effect refers to the effect achieved when the active ingredients are used together being greater than the sum of the effects produced by using the compounds individually; the additive effect refers to the effect achieved when the active ingredients are used together being equivalent to the sum of the effects produced by using the compounds individually.

[0017] Preferably, the other therapeutic agents are helpful for the treatment of osteosarcoma. These other therapeutic agents may be selected from at least one of cisplatin, doxorubicin, doxorubicin, mesna, ifosfamide, dacarbazine, and methotrexate.

[0018] Furthermore, this invention has discovered that the combined use of schisandrin B and cisplatin can enhance the effect of inhibiting tumor growth and reducing tumor volume, while also reducing toxic side effects, indicating a synergistic effect between the two.

[0019] Therefore, the pharmaceuticals and pharmaceutical compositions described in this invention may further comprise other therapeutic agents. Preferably, the other therapeutic agents are selected from cisplatin.

[0020] In one embodiment of the invention, a pharmaceutical composition comprising schisandrin B and cisplatin is provided. Preferably, the pharmaceutical composition is used to treat osteosarcoma.

[0021] The weight ratio of schisandrin B to cisplatin is 5 to 40:1, preferably 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, or 40:1; more preferably, it is 10 to 30:1, and even more preferably, it is 15 to 20:1.

[0022] In one embodiment of the invention, the invention also provides the use of the pharmaceutical composition in the preparation of a medicament for treating osteosarcoma. Preferably, the medicament is used to inhibit the proliferation and growth of osteosarcoma cells and / or inhibit the migration and invasion of osteosarcoma cells and / or induce osteosarcoma cell apoptosis. More preferably, the induction of osteosarcoma cell apoptosis is achieved by enhancing osteosarcoma cell autophagy.

[0023] In one embodiment of the invention, the invention also provides a pharmaceutical combination product comprising schisandrin B and cisplatin.

[0024] The weight ratio of schisandrin B to cisplatin is 5 to 40:1, preferably 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, or 40:1; more preferably, it is 10 to 30:1, and even more preferably, it is 15 to 20:1.

[0025] Preferably, schisandrin B and cisplatin are present as separate compositions.

[0026] Preferably, at least one of the schisandrin and cisplatin compositions contains a pharmaceutically acceptable excipient. More preferably, both the schisandrin and cisplatin compositions contain a pharmaceutically acceptable excipient.

[0027] The drug combination products can be administered simultaneously or sequentially. When administered sequentially, the combination can be administered in two or more ways. Schisandra chinensis and cisplatin can be administered together in a single drug composition or separately in different drug compositions, and when administered separately, they can be administered simultaneously or sequentially in any order.

[0028] In one embodiment of the invention, the invention also provides the use of the pharmaceutical combination product in the preparation of a medicament for treating osteosarcoma. Preferably, the medicament is used to inhibit the proliferation and growth of osteosarcoma cells and / or inhibit the migration and invasion of osteosarcoma cells and / or induce osteosarcoma cell apoptosis. More preferably, the induction of osteosarcoma cell apoptosis is achieved by enhancing osteosarcoma cell autophagy.

[0029] The present invention has the following beneficial effects:

[0030] 1. This invention has discovered that schisandrin B has a very outstanding effect in the treatment of osteosarcoma. It can not only effectively inhibit the growth and reproduction of osteosarcoma cells and induce osteosarcoma cell apoptosis, but also inhibit their migration and invasion. This has a very outstanding therapeutic effect on osteosarcoma, a primary, invasive and easily metastatic malignant tumor. Animal experiments show that it can greatly reduce the volume of osteosarcoma.

[0031] 2. Schisandrin B has low toxicity, which can reduce damage to patients and minimize toxic side effects when used in treatment.

[0032] 3. Schisandrin B exhibits a synergistic effect when used in combination with drugs such as cisplatin. On one hand, schisandrin B can significantly enhance the therapeutic effect of cisplatin, achieving a synergistic effect; on the other hand, schisandrin B not only has low toxicity but can also reduce the toxic side effects of cisplatin, thus protecting patients when used in combination with cisplatin. Therefore, the combined use of schisandrin B and cisplatin has a synergistic effect and reduces toxicity. Attached Figure Description

[0033] Figure 1 Effect of schisandrin B on the growth activity of osteosarcoma cells: CCK8 assay;

[0034] Figure 2 To investigate the effects of schisandrin B on the migration and invasion abilities of osteosarcoma cells: (A) scratch assay; (B) Transwell assay; (C) and (D) statistical analysis;

[0035] Figure 3 The effect of schisandrin B on osteosarcoma cell apoptosis: (A) Western blot (WB) experiment; (B) statistical analysis; (C) flow cytometry; (D) TUNEL staining.

[0036] Figure 4 Schisandrin enhances autophagy in osteosarcoma cells: (A) Western blot results; (B) statistical analysis; (C) and (D) immunofluorescence detection.

[0037] Figure 5 The tumor formation status and tumor volume in nude mice in the schisandrin B group. Detailed Implementation

[0038] The following will describe preferred embodiments of the invention in detail. These embodiments are provided to better illustrate the invention and are not intended to limit the invention to these examples. Non-essential improvements and adjustments to the embodiments based on the invention's description still fall within the scope of the invention.

[0039] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Where specific techniques or conditions are not specified in the embodiments, they should be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions.

[0040] Example 1: Schisandrin inhibits the growth, migration, and invasion of osteosarcoma cells.

[0041] Experimental Procedure

[0042] 1-1CCK8 assay: Inhibition of cell proliferation

[0043] step:

[0044] 1. Take a new 96-well plate and seed human osteosarcoma cells MG-63 (Otwo Biotech, HTX1630) into each well, at a ratio of 1 x 10⁶ cells / well. 4 100 μl per well, 5-6 replicates per group;

[0045] 2. Place the seeded 96-well plate in a 37°C, 5% CO2 incubator overnight for cell adhesion.

[0046] 3. After the cells adhere to the culture medium, remove the old culture medium and add 100 μl of different concentrations of schisandrin (0, 1, 2, 5, 10, 20, 40, 80 and 160 μg / ml) of culture medium containing 1% serum to each well according to the group. A blank control group (containing culture medium but no cells) is also set up.

[0047] 4. Continue incubation in the incubator for 24 hours;

[0048] 5. After the cell stimulation time is over, add 10 μl of CCK8 solution to each well (be careful not to generate bubbles) and stimulate for 1-4 hours;

[0049] 6. After the stimulation time is over, the absorbance (OD) value at 450 nm is measured using an ELISA reader, and cell viability is calculated based on the OD value.

[0050] 1-2 Scratch assay: Inhibition of cell migration ability

[0051] step:

[0052] 1. Place all necessary instruments into the laminar flow hood for UV sterilization for 30 minutes;

[0053] 2. First, use a marker pen to draw horizontal lines evenly on the back of the 6-hole board, using a ruler as a guide. Draw one line approximately every 0.5-1cm, and make sure each hole has at least 5 lines.

[0054] 3. Digest human osteosarcoma cells MG-63 and seed them into 6-well plates, approximately 5 × 10⁶ cells per well. 5 200 μl of cells were seeded and placed in an incubator overnight to allow them to adhere to the cell wall.

[0055] 4. After the cells adhere to the plate on the second day, take out the 6-well plate and use a 10μl pipette tip, holding it as perpendicular as possible to the horizontal line on the back of the plate, with the pipette tip vertical and not tilted.

[0056] 5. After the scratching process, wash the cells three times with PBS to remove the scratched cells. Add schisandrin B prepared with serum-free culture medium (0, 20 μg / ml, 40 μg / ml) according to the groups, and take pictures (0h).

[0057] 6. Place in an incubator and continue culturing. Change the medium every other day. Take samples at 24h, 48h and 72h, take photos and calculate the migration rate.

[0058] 1-3 Transwell Experiment: Inhibition of Cell Invasion

[0059] step:

[0060] 1. Human osteosarcoma cells MG-63 were cultured in serum-free medium for 24 hours in advance to eliminate the influence of serum;

[0061] 2. Take out the matrix gel in advance and place it at 4 degrees Celsius. Use a 1:8 dilution of 50 mg / L matrix gel to coat the upper surface of the bottom membrane of the Transwell chamber, avoiding the formation of air bubbles. Place it in an incubator and wait for it to air dry.

[0062] 3. Remove MG-63 cells that have been starved for 24 hours, digest them with trypsin, centrifuge to remove the culture medium after digestion, resuspend the cells in serum-free culture medium, and adjust the cell concentration to 5 × 10⁻⁶. 5 / ml;

[0063] 4. Add 100 μl of cell suspension to the upper chamber of Transwell and group them according to the experimental requirements. Different concentrations of schisandrin (20 μg / ml, 40 μg / ml) were added to the experimental groups.

[0064] 5. Add 600 μl of culture medium containing 10% serum to the lower chamber of the Transwell (bubbles may be generated between the lower culture medium and the chamber; once bubbles are generated, lift the chamber, remove the bubbles, and then place the chamber back into the culture plate);

[0065] 6. Place the completed Transwell plate into an incubator and incubate for 24 hours;

[0066] 7. After the incubation period is over, remove the chamber, aspirate the liquid from the upper chamber, and transfer it to a well containing approximately 800 μl of pre-cooled methanol. Fix at room temperature for 30 min.

[0067] 8. After the fixation time is over, aspirate the fixative from the upper chamber and transfer it to a well in which approximately 800 μl of crystal violet has been added. Stain at room temperature for 15-30 min.

[0068] 9. After staining, gently soak and wash several times with PBS, remove the chamber, aspirate the liquid in the upper chamber, and carefully wipe the unpenetrated cells on the bottom membrane surface of the upper chamber with a damp cotton swab;

[0069] 10. Place the chamber on a glass slide and observe nine random fields of view under a microscope, then statistically analyze the results.

[0070] 1-4WB Experiments: Exploring Mechanisms

[0071] step:

[0072] 1. Take out MG-63 cells treated with schisandrin B (0 and 20 μg / ml, 40 μg / ml) for 24 h, aspirate the culture medium, wash the cells 3 times with pre-cooled PBS, digest the cells with trypsin, collect them into centrifuge tubes, centrifuge at 2000 rpm for 5 min, remove the supernatant, add 1 ml of PBS to each well, and take 20 μl for cell counting.

[0073] 2. Prepare cell lysis buffer: Add 10 μl of phosphatase inhibitor, 1 μl of protease inhibitor and 10 μl of PMSF to 1 ml of cell lysis buffer, mix well and store on ice.

[0074] 3. Divide the counted cells into groups of 10. 6 Add cell lysis buffer at a ratio of 100 μl per cell;

[0075] 4. After adding the lysis buffer, place on ice for lysis for 30 minutes, and blow air through the ice every 10 minutes;

[0076] 5. After the lysis time is complete, add 5x loading buffer at a ratio of lysis buffer: 5x loading buffer = 4:1;

[0077] 6. Preheat the water bath to 100℃ and cook the egg whites for 10 minutes. After cooking, store the egg whites at -20℃.

[0078] 7. Clean the glass plate and prepare a 10% adhesive solution according to the instructions.

[0079] 8. Load the extracted proteins sequentially (Ctrl, 20μg / ml, 40μg / ml) for electrophoresis. Select an electrophoresis voltage of 80V and a time of approximately 2-3 hours.

[0080] 9. Activate the cut PVDF membrane in methanol for more than 10 seconds in advance. Transfer the membrane in the following order: black-filter paper-gel-membrane-filter paper-white. Select a transfer current of 300mA and a transfer time of about 90 minutes.

[0081] 10. After the transfer is complete, seal with 5% skim milk powder at room temperature for 1-2 hours;

[0082] 11. After the blocking time is over, prepare the required primary antibody at a ratio of 1:1000, cut the membrane according to the molecular weight, and incubate the primary antibody overnight at 4°C.

[0083] 12. Collect the primary antibody on the second day, wash three times with 1% TBST for 10 minutes each time;

[0084] 13. Prepare the secondary antibody at a ratio of 1:10000, and prepare the corresponding secondary antibody according to the requirements of the primary antibody. Incubate the secondary antibody at room temperature for 1 hour.

[0085] 14. After the secondary antibody incubation time is over, wash three times with 1% TBST, 10 minutes each time;

[0086] 15. Finally, prepare the developer solution at a 1:1 ratio, expose it on the exposure machine, and save the results.

[0087] 1-5 Tunel experiments: Increased apoptosis

[0088] step:

[0089] 1. Seed healthy MG-63 cells onto 24-well slides, approximately 1 x 10⁶ cells per well. 5 After the cells adhered to the wall, they were stimulated with schisandrin B (0, 20 μg / ml, 40 μg / ml) for 24 hours.

[0090] 2. After the stimulation time is over, wash with PBS 3 times, 3 minutes each time;

[0091] 3. Prepare 4% paraformaldehyde, fix the cells for 30 min, and then wash them again with PBS;

[0092] 4. Next, add PBS containing 0.3% Triton X-100 and incubate at room temperature for 5 minutes to permeate the cells;

[0093] 5. Prepare the detection solution for the one-step Tunel cell apoptosis detection kit according to the instructions (C1086, Beyotime);

[0094] 6. After the permeation time of the slides is completed, wash them twice with PBS, then add 50 μl of TUNEL detection solution to each slide and incubate at 37°C in the dark for 60 min. Note: 50 μl of TUNEL detection solution is suitable for smears, sections, or one well of a 96-well, 48-well, 24-well, or 12-well plate. If it is one well of a 6-well plate, 100 μl of TUNEL detection solution should be used.

[0095] 7. After the incubation period, wash the slide three times with PBS, add 20 μl of anti-fluorescence quenching mounting solution containing DAPI, mount the slide, and observe and photograph it under a fluorescence microscope.

[0096] 1-6 Immunofluorescence: Exploring Cellular Mechanisms

[0097] step:

[0098] 1. In a culture plate, MG-63 cells that have been stimulated with schisandrin B (0, 20 μg / ml, 40 μg / ml) for 24 h were placed on a slide and the slide was washed with PBS 3 times for 3 min each time.

[0099] 2. Fix the slide with 4% paraformaldehyde for 15 min, and wash the slide with PBS 3 times, 3 min each time;

[0100] 3. 0.5% Triton X-100 (prepared with PBS) permeate at room temperature for 20 min;

[0101] 4. Wash the slide with PBS 3 times for 3 minutes each time, blot dry with absorbent paper, add normal goat serum to the slide, and block at room temperature for 30 minutes.

[0102] 5. Absorb the blocking solution with absorbent paper, do not wash, add a sufficient amount of diluted primary antibody to each slide and place it in a humidified chamber, incubate overnight at 4°C;

[0103] 6. Day 2: Add fluorescent secondary antibody: Wash the slides with PBST 3 times, 3 minutes each time. After blotting off the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody. Incubate in a humidified chamber at 20-37℃ for 1 hour. Wash the slides with PBST 3 times, 3 minutes each time. Note: From the time the fluorescent secondary antibody is added, all subsequent operations should be performed in a dark place as much as possible.

[0104] 7. Counterstaining the nucleus: Add DAPI and incubate in the dark for 5 min to stain the nucleus. Wash away excess DAPI by washing with PBST for 5 min × 4 times.

[0105] 8. Blot the liquid off the slide with absorbent paper, seal the slide with mounting solution containing anti-fluorescence quencher, and then observe the acquired image under a fluorescence microscope.

[0106] Flow cytometry cell counting experiments 1-7: Increased apoptosis

[0107] step:

[0108] 1. Cell collection: Supernatant from MG-63 cells in different groups stimulated with schisandrin B (0, 20 μg / ml, 40 μg / ml) for 24 h was collected into 15 ml EP tubes. The cells were washed with 2 ml PBS, and 1 ml of trypsin without EDTA was added. The cells were digested in a 37°C incubator until large cell fragments detached from the bottom of the dish. After digestion was stopped, the cells were transferred to 15 ml EP tubes, centrifuged at 2000 rpm at room temperature for 5 min, and the supernatant was discarded.

[0109] 2. Wash and resuspend cells with 1 ml of room temperature PBS, and count cells using a cell counter. Take 2 x 10⁻⁶ cells from each group. 6 Cells were transferred to 1.5 ml EP tubes, centrifuged at 2000 rpm for 5 min at room temperature, and the supernatant was discarded.

[0110] 3. Staining: Resuspend cells (2 x 10⁻⁶) in 200 μl of buffer. 6All experimental groups and the negative control group require double staining, while the positive control requires single-staining tubes with two dyes. Add 5 μl Annexin V dye and 5 μl 7-AAD to the double-staining tubes, and add either 5 μl Annexin V dye or 5 μl 7-AAD to the single-staining tubes. Gently smear by hand, avoiding staining for 15 minutes (during staining, prepare flow cytometry tubes and filter membranes, turn on the flow cytometer, and check the sheath fluid and waste fluid).

[0111] 6. After staining, add 1 ml PBS, centrifuge at 2000 rpm at room temperature for 5 min, discard the supernatant, resuspend in 100 μl PBS, and filter through a 200 mesh filter.

[0112] 7. Go to the computer and record the experimental results.

[0113] Experimental Results

[0114] 1. Schisandrin B inhibits the growth activity of osteosarcoma cells MG-63.

[0115] First, osteosarcoma cells MG-63 were stimulated with schisandrin B at concentrations of 0, 1, 2, 5, 10, 20, 40, 80, and 160 μg / ml. The effect on cell growth activity was then assessed using the CCK8 assay. The results are as follows: Figure 1 As shown, the activity of MG-63 cells gradually decreased with increasing schisandrin concentration, exhibiting a concentration-dependent effect. When the schisandrin concentration was 20 μg / ml and 40 μg / ml, the cell viability was approximately 60%, and this concentration was selected as the stimulation concentration for subsequent experiments. * indicates P < 0.05vsCtrl.

[0116] 2. Schisandrin B inhibits the migration and invasion abilities of osteosarcoma cells MG-63.

[0117] To investigate the effect of schisandrin B on the migration ability of osteosarcoma cells, osteosarcoma cells were stimulated with different concentrations (20 μg / ml and 40 μg / ml) of schisandrin B, followed by a scratch assay. The results are as follows: Figure 2 As shown in Figure A, compared with the control group, after 72 hours of stimulation with schisandrin B, the ability of cells to migrate towards the center was weakened, and the higher the concentration, the weaker the migration ability. Statistical analysis indicated that this was statistically significant. Figure 2 C). Where * indicates P < 0.05 vs Ctrl.

[0118] To further verify the effect of schisandrin B on the invasive ability of osteosarcoma cells, osteosarcoma cells were stimulated with different concentrations (20 μg / ml and 40 μg / ml) of schisandrin B and then subjected to Transwell assays. The experimental results are as follows: Figure 2As shown in Figure B, stimulation with schisandrin significantly reduced the number of osteosarcoma cells migrating downwards within the commissure chambers, and this reduction exhibited a certain concentration-dependent effect. Statistical analysis was performed. Figure 2 D) This indicates statistical significance. The above results show that schisandrin B can inhibit the migration and invasion of osteosarcoma cells. * indicates P < 0.05 vs Ctrl.

[0119] 3. Schisandrin B induces apoptosis in osteosarcoma cells MG-63.

[0120] To further confirm that schisandrin B can induce apoptosis in osteosarcoma cells, osteosarcoma cells were first stimulated with different concentrations (20 μg / ml and 40 μg / ml) of schisandrin B, followed by Western blotting. The results are as follows: Figure 3 As shown in Figure A, the expression levels of pro-apoptotic proteins Bax and caspase 3 increased, while the expression levels of anti-apoptotic proteins Bcl-2 and PNCA decreased, and the results were statistically significant. Figure 3 B) The results were also verified by flow cytometry. Figure 3 Flow cytometry results showed that stimulation of osteosarcoma MG-63 cells with schisandrin increased apoptosis. Finally, TUNEL staining further validated these results. Figure 3 As shown in Figure D, schisandrin B significantly increased green fluorescence in osteosarcoma cells after stimulation. All the above experimental results indicate that schisandrin B can induce apoptosis in osteosarcoma cells. * indicates P < 0.05 vs Ctrl.

[0121] 4. Schisandrin B induces apoptosis in osteosarcoma cells MG-63 by enhancing autophagy.

[0122] To verify the mechanism by which schisandrin induces apoptosis in osteosarcoma cells MG-63, Western blotting was used to detect the expression levels of autophagy-related proteins LC3, P62, ATG5, and Beclin1. The results are as follows: Figure 4 As shown in Figure A, stimulation of osteosarcoma cells with schisandrin B resulted in upregulation of autophagy-related proteins LC3II / LC3I, ATG5, and Beclin 1, and downregulation of P62. Statistical analysis of these proteins showed significant differences. Immunofluorescence assays were used to detect the expression levels of LC3II and P62. Figure 4 C and Figure 4 D) Consistent with WB, LC3II expression increased while P62 expression decreased. All these results confirm that schisandrin can induce apoptosis in osteosarcoma cells MG-63 by enhancing autophagy. * indicates P < 0.05 vs Ctrl.

[0123] Example 2: Schisandra chinensis combined with cisplatin treatment

[0124] Experimental Procedure

[0125] Osteosarcoma model construction and grouping: Purchase 4-6 week old male Balb / c nude mice (weighing approximately 15-20g), culture osteosarcoma cells MG-63, transfect with the eukaryotic expression recombinant plasmid pRc / CMV2-luc carrying the luciferase reporter gene, and select using G418 to obtain cell clones stably expressing the luciferase gene. After the cells are in good growth condition, collect the cells at a density of approximately 1×10⁻⁶. 7 Mix 100 μl of each tumor with 100 μl of serum-free DMEM medium, for a total of 200 μl, and inject subcutaneously into nude mice. Observe and record the changes in tumor size daily. When the subcutaneous tumor size reaches 200 mm... 3 Nude mice that successfully developed the tumor model were randomly divided into four groups. The control group was given an equal volume of saline by gavage. Schisandra chinensis B was administered to the nude mice at a concentration of 20 mg / kg / day by gavage. Cisplatin was administered at a concentration of 1 mg / kg / day by intraperitoneal injection. Tumor volume and mouse weight were recorded every three days. After one month, mice were subjected to in vivo imaging and X-ray imaging to observe for distant metastasis. Mice were then sacrificed, subcutaneous tumors were dissected, and tissue samples were taken for pathological sectioning and other related experiments.

[0126] Experimental Results

[0127] 1. After treatment with schisandrin B combined with cisplatin, the general condition of nude mice with osteosarcoma was significantly better than that of the group treated with cisplatin alone.

[0128] Because the main side effects of high-dose cisplatin treatment are gastrointestinal discomfort and kidney damage, nude mice in different groups were treated with different methods for 30 days. Their mental state, activity level, appetite, and bowel and bladder function were then observed and statistically analyzed. The results are shown in Table 1 below. After cisplatin treatment alone, the nude mice showed poor mental state, reduced activity, and decreased appetite. Observation of their stool and urine revealed blood in the stool and reduced urine output. However, treatment with schisandrin B combined with cisplatin improved the mental state and appetite of the nude mice, and alleviated bowel and bladder function issues. This confirms that schisandrin B can alleviate the side effects caused by cisplatin treatment.

[0129] Table 1: Summary of basic conditions of nude mice in different treatment groups at 30 days

[0130]

[0131] 2. Treatment with schisandrin B combined with cisplatin resulted in smaller osteosarcoma tumors and a higher survival rate in nude mice compared to treatment with cisplatin alone.

[0132] After 30 days of treatment in different groups of nude mice, the changes in tumor size and survival were observed in each group. For example, the schisandrin B group... Figure 5As shown in Table 2, the survival rate of nude mice was statistically analyzed after excluding deaths caused by other reasons. The results are shown in Table 2 below. Tumor volume (V) = 1 / 2ab2 (a is the major axis, b is the minor axis).

[0133] Table 2: Tumor size and survival in nude mice under different treatment groups at 30 days

[0134]

[0135] As shown in the table above, cisplatin alone reduced tumor size in nude mice compared to the control group, but the mortality rate was high. Combined treatment with schisandrin and cisplatin resulted in more significant tumor size reduction and a lower mortality rate compared to cisplatin alone or cisplatin alone. These experimental results suggest that schisandrin combined with cisplatin can significantly inhibit tumor growth in osteosarcoma and improve the survival rate of nude mice. Using the formula q = P A+B / (P A +P B -P A ×P B The calculated synergy index is 1.45 (P). A P represents the treatment rate of cisplatin. B The therapeutic rate of schisandrin B is represented by P. A+B The treatment rate of cisplatin + schisandrin B is expressed as (tumor volume before treatment - tumor volume after treatment) / tumor volume before treatment. q < 1 indicates that the two drugs have an antagonistic effect when used together; q > 1 indicates that the two drugs have a synergistic effect when used together; q = 1 indicates that the two drugs have an additive effect when used together. It has a very significant synergistic effect.

[0136] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of osteosarcoma, the pharmaceutical composition comprising schisantherin B and cisplatin, wherein the weight ratio of schisantherin B and cisplatin is 20:

1.

2. Use of a pharmaceutical combination product in the manufacture of a medicament for the treatment of osteosarcoma, the pharmaceutical combination product comprising schisantherin B and cisplatin, wherein the weight ratio of schisantherin B and cisplatin is 20:

1.

3. Use according to claim 2, characterized in that, Schisantherin B and cisplatin are administered separately in different pharmaceutical compositions, and when administered separately, simultaneously or sequentially in any order.

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